Resistin and Visfatin Expression in HCT-116 Colorectal Cancer Cell Line.

Adipocytokines, hormones secreted from adipose tissue, have been shown to be associated with many cancers such as breast, prostate and colorectal cancer. Recent studies have indicated that resistin and visfatin, two of these adipokines have high level plasma concentrations in colorectal cancer patients and may be promising biomarkers for colorectal cancer. The aim of this study was to identify whether the colorectal cancer cell line, HCT-116, itself is the source of these two adipokines secretion. Resistin and visfatin expression were investigated in HCT-116 by RT - PCR at mRNA level and confirmed by ELISA at protein level. Visfatin showed a high expression at both mRNA and protein levels in HCT-116. Conversely, resistin was not expressed in either cell lysate or supernatant. These results showed that HCT-116 colorectal cancer cells secrete and express visfatin endogenously. However, they are not the main source of resistin and the high level of resistin in colorectal cancer may be due to monocytes and other inflammatory cells which increase in proinflammation status of cancer. Taken together, visfatin may act on colorectal cancer cell in an autocrine manner while resistin may act in a paracrine manner.

cytoplasm and mitochondria and extracellular (eNampt) forms (22)(23). Since it was initially described as a growth factor for early B cell proliferation isolated from peripheral blood lymphocytes, PBEF (pre-B-cell colony-enhancing factor) is also used to refer to this protein (24).
Visfatin has been shown to have insulin mimetic effect and plays many pathophysiologic conditions, including metabolism, immune response and cancer (25)(26). Visfatin up-regulation has been reported in many inflammatory related disorders such as atherosclerosis, inflammatory bowel disease, psoriasis, rheumatoid arthritis, osteoarthritis, acute lung injury and chronic obstructive pulmonary disease (14). There are also several documents revealing visfatin expression in many malignant tissues including prostate, breast, endometrium and glioblastoma (27)(28)(29)(30)). Although high visfatin plasma level has been found in colorectal cancer patients, the ability of colorectal cancer cell to express this adipokine and the main cause of its high plasma concentration is still unclear.
To clarify the colorectal cancer cell potential to secrete these two adipokines, we looked for

Cell culture
Human HCT-116 colorectal epithelial cancer cells were bought from Pasteur Institute Cell Bank (IRI) and grown in high glucose DMEM medium (Invitrogen, USA) supplemented with 10% fetal bovine serum (Biochem, Germany), penicillin/streptomycin (100U/ml and 100 mg/ml, respectively) at 37˚C, under 5% CO 2 atmosphere. The cells were seeded at 1.5×10 6 cells in 25-T culture flasks and allowed to attach overnight in an incubator. The total medium was then replaced with serum-free medium for 24 h and 48h to be ensured about the absence of any similar factors in the medium disturbing these adipokines study in ELISA method and allow the cells to secrete fresh proteins. Since intracellular proteins are isolated from the medium and there is no confusing factor for adipokine study in cell lysate samples, serum-free medium replacement was not done for such samples.
Therefore, the cells were harvested after overnight incubation to achieve the same cell counts (1.5×10 6 ).

Cell preparation and ELISA
The supernatant of HCT-116 cells was

Results
To determine the visfatin and resistin expression and secretion from colorectal cancer   As the aim of this study was to find whether HCT-116 cell lines are able to express these two adipokines and not to compare its ability with the normal cells, no other cell culture was investigated for the presence of these mRNAs. Figure 1 shows the RT-PCR results of resistin and visfatin genes.
Visfatin showed a sharp band at 189 bp, however, cDNA band of resistin was not detected on gel.
GAPDH primers were used in RT-PCR as internal control.

Discussion
Despite the fact that adipose tissue is a primary source of adipokines and obesity, the condition accompanied with high serum levels of adipokines and related to many malignancies including colorectal cancer (31)(32)(33)(34), recent investigation has demonstrated that resistin and visfatin plasma levels are significantly increased in colorectal cancer (CRC) patients independent of BMI and may be good biomarkers for malignant potential and stage progression of this cancer (13,32). However, the other major sources of these two adipokines may also be responsible for their high serum concentrations in CRC patients rather than adipose tissue. To date, there are only a few documents about resistin and visfatin expression in CRC (15,(35)(36).